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AIM Cardiac pathologies are accompanied by alterations in substrate metabolism, and extracellular flux analysis is a standard tool to investigate metabolic disturbances, especially in immortalized cell lines. However, preparations of primary cells, such as adult cardiomyocytes require enzymatic dissociation and cultivation affecting metabolism. Therefore, we developed a flux analyzer-based method for assessment of substrate metabolism in intact vibratome-sliced mouse heart tissue. METHODS Oxygen consumption rates were determined using a Seahorse XFe24-analyzer and "islet capture plates". We demonstrate that tissue slices are suitable for extracellular flux analysis, and metabolize both free fatty acids (FFA) and glucose/glutamine. Functional integrity of tissue slices was proven by optical mapping-based assessment of action potentials. In a proof-of-principle approach, the sensitivity of the method was tested by analyzing substrate metabolism in the remote myocardium after myocardial infarction (I/R). RESULTS Here, I/R increased uncoupled OCR compared to sham-animals indicating a stimulated metabolic capacity. This increase was caused by a higher glucose/glutamine metabolism, whilst FFA oxidation was unchanged. CONCLUSION In conclusion, we describe a novel method to analyze cardiac substrate metabolism in intact cardiac tissue slices by extracellular flux analysis. The proof-of-principle experiment demonstrated that this approach has a sensitivity allowing the investigation of pathophysiologically relevant disturbances in cardiac substrate metabolism.